Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway.

doi: 10.4049/jimmunol.1303275

Figure Lengend Snippet: FIGURE 3. MSA inhibits ULBP2 protein surface transport. (A) Jurkat Tag- 9 cells were transiently transfected with the ULBP2-myc-GFP construct. Two hours posttransfection cells were left untreated or treated with either 20 ng/ml FR901228, 5 mM MSA, or a combination of both. After 18 h cells were analyzed for myc-tag cell surface expression by flow cytometry. Cells were gated according to nontransfected cells. (B) Jurkat Tag-9 cells were transiently transfected with the ULBP2-myc-GFP construct. Two hours posttransfection cells were left untreated or treated with various concentrations of MSA (5 or 10 mM). After 18 h cells were lysed and used for Western blotting against GFP (top, molecular mass of ∼64 kDa) and ERK1 (bottom, molecular mass of 44 kDa). (C) Jurkat Tag-9 cells were transiently transfected with the ULBP2-myc- GFP construct. Two hours posttransfection cells were left untreated or treated with 5 mM MSA. After 18 h, cell images were recorded using a Zeiss Cel- lObserver equipped with a Yokogawa CSU-X1 spinning disk acquiring full Z- stacks of the cells in the EGFP channel using an alpha Plan-Apochromat oil 3100/1.46 numerical aperture objective. Z-stacks at position 36 were chosen to compare construct location in untreated cells (left top) and MSA-treated cells (right top). Full Z-stacks were used to create three-dimensional images of the construct location in untreated cells (left bottom) and MSA-treated cells (right bottom). Scale bar, 2 mm. Data are representative of four separate experiments [(A), means 6 SD] or two experiments (B and C). **p , 0.01.

Article Snippet: The Abs used for Western blotting were biotinylated anti–human ULBP2 (R&D Systems, BAF1298), anti-ERK1 (Santa Cruz Biotechnology, sc-93), anti-CD74/Ii (Novus Biologicals, NBP1-33109), anti-GAPDH (Fitzgerald, 10R-G109a), streptavidin-peroxidase polymer (ultrasensitive) (Sigma-Aldrich, 2438), peroxidase-conjugated rabbit anti-mouse Igs (Dako, P0260), and peroxidase-conjugated swine antirabbit Igs (Dako, P0399).

Techniques: Transfection, Construct, Expressing, Cytometry, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway.

doi: 10.4049/jimmunol.1303275

Figure Lengend Snippet: FIGURE 5. MSA is able to inhibit the production of soluble ULBP2 in primary melanoma cells. (A) FM-78, FM-86, and SK-MEL28 were left untreated or either treated with 20 ng/ml FR901228 or 10 mM MSA alone or in combination. After 18 h cells were analyzed for MICA/B and ULBP2 surface expression by flow cytometry. (B) FM-78 (top), FM-86 (middle), and SK-MEL28 (bottom) cells were left untreated or treated with either 20 ng/ml FR901228 or 10 mM MSA alone or in combination for 18 h using culture medium containing 0.5% FBS. The proteins in the media fraction were precipitated by TCA as described in Materials and Methods. Western blotting of precipitates was performed using an Ab against ULBP2 (top; molecular mass of 35 kDa). Serum proteins (SP) (bottom; molecular mass

Article Snippet: The Abs used for Western blotting were biotinylated anti–human ULBP2 (R&D Systems, BAF1298), anti-ERK1 (Santa Cruz Biotechnology, sc-93), anti-CD74/Ii (Novus Biologicals, NBP1-33109), anti-GAPDH (Fitzgerald, 10R-G109a), streptavidin-peroxidase polymer (ultrasensitive) (Sigma-Aldrich, 2438), peroxidase-conjugated rabbit anti-mouse Igs (Dako, P0260), and peroxidase-conjugated swine antirabbit Igs (Dako, P0399).

Techniques: Expressing, Cytometry, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway.

doi: 10.4049/jimmunol.1303275

Figure Lengend Snippet: FIGURE 6. ULBP2 induction is dependent on PKC activation. (A) Jurkat E6-1 cells were left untreated or incubated with 3 mM PMA or 300 mM ingenol. After 18 h cells were analyzed for MICA/B and ULBP2 surface expression by flow cytometry. (B) Jurkat E6-1 cells were left untreated or treated once, twice, or four times with 20 mM ingenol-3- angelate during a time period of 16 h. After incubation cells were ana- lyzed for ULBP2 surface expression by flow cytometry. (C) Jurkat E6-1 cells were treated for the indicated time points with 20 ng/ml FR901228 or 3 mM PMA. At the indicated time points cells were analyzed for MICA/B and ULBP2 cell surface expression by flow cytometry. (D) Jurkat E6-1 cells were left untreated or treated with 3 mM PMA or 20 ng/ ml FR901228 alone or in combination with 1 mM of the PKC inhibitors Go¨6983 (left) or Go¨6976 (right). The PKC inhibitors were added to the cell culture for the first time together with FR901228 or PMA and for the second time 4–6 h later. After 18 h, cells were analyzed for ULBP2 surface expression by flow cytometry. (E) Jurkat E6-1 cells were left untreated or treated with 3 mM PMA alone or in combination with either 5 or 10 mM MSA. After 18 h, cells were analyzed for MICA/B and ULBP2 surface expression by flow cytometry. Data are representative of

Article Snippet: The Abs used for Western blotting were biotinylated anti–human ULBP2 (R&D Systems, BAF1298), anti-ERK1 (Santa Cruz Biotechnology, sc-93), anti-CD74/Ii (Novus Biologicals, NBP1-33109), anti-GAPDH (Fitzgerald, 10R-G109a), streptavidin-peroxidase polymer (ultrasensitive) (Sigma-Aldrich, 2438), peroxidase-conjugated rabbit anti-mouse Igs (Dako, P0260), and peroxidase-conjugated swine antirabbit Igs (Dako, P0399).

Techniques: Activation Assay, Incubation, Expressing, Cytometry, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway.

doi: 10.4049/jimmunol.1303275

Figure Lengend Snippet: FIGURE 7. The lysosomal/endosomal transport pathway is important for ULBP2 cell surface expression and is facilitated by invariant chain. (A) FR901228-treated Jurkat E6-1 cells with lysosomal inhibitors (from left to right: ammonium chloride, bafilomycin A1, chloroquine) at indicated concentrations. Cells were analyzed for MICA/B and ULBP2 surface expression. (B) Jurkat Tag-9 cells were transiently transfected with either the ULBP2-myc-GFP (top) or MICA*018-myc-GFP (bottom) construct and treated with 10 mM chloroquine for 18 h. For labeling and tracking of the acetic organelles, cells were stained as described in Materials and Methods. Images were taken in two channels: enhanced GFP (EGFP; green) and Lyso- Tracker Red DND-99 (red). Colocalization is shown in yellow. Scale bar, 2 mm. (C) FR901228-treated FM-86 cells with 10 and 30 nM bafilomycin A1. Cells were analyzed for MICA/B and ULBP2 surface expression. (D) Jurkat Tag-9 cells were transfected with two different siRNAs against CD74 or their corresponding siRNA control. Six hours posttransfection cells were treated with 20 ng/ml FR901228. After 18 h half of the cells were analyzed for ULBP2 (left), MICA/B (middle), and ICAM-I (right) surface expression. The remaining cells were lysed and used for Western blotting against Ii (molecular mass of ∼43 kDa) and GAPDH (molecular mass of 37 kDa). (E) Jurkat Tag-9 cells and FM-86 cells were transfected with an Ii construct or a control plasmid. After 24 h cells were treated with FR901228 or kept untreated for 18 h. Cells were analyzed for MICA/B and ULBP2 surface expression. (F) IFN-g–stimulated (0.5 or 5 ng/ml) and nonstimulated monocytes were left untreated or treated with 10 mM MSA (18 h). Cells were analyzed for MHC class II surface expression. Data are representative of at least three separate experiments [(A) and (C)–(F), means 6 SD] or two separate experiments (B). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.001.

Article Snippet: The Abs used for Western blotting were biotinylated anti–human ULBP2 (R&D Systems, BAF1298), anti-ERK1 (Santa Cruz Biotechnology, sc-93), anti-CD74/Ii (Novus Biologicals, NBP1-33109), anti-GAPDH (Fitzgerald, 10R-G109a), streptavidin-peroxidase polymer (ultrasensitive) (Sigma-Aldrich, 2438), peroxidase-conjugated rabbit anti-mouse Igs (Dako, P0260), and peroxidase-conjugated swine antirabbit Igs (Dako, P0399).

Techniques: Expressing, Transfection, Construct, Labeling, Staining, Control, Western Blot, Plasmid Preparation

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A, B) NK cell-mediated specific down-regulation of ULBP2. 10 5 Jurkat (left panels) or H9 cells (right panels) were incubated with (+NK) or without (−NK) in an equal number of IL-2 expanded peripheral blood NK cells at 37°C for 2 hours. The resulting cell mixtures were stained by anti-human MICA/B, ULBP1, ULBP2 or ULBP3 antibodies and analyzed by flow cytometry (solid lines). NK cells were excluded by anti-human CD56 mAb staining. Isotype controls are shown in gray-shaded histograms. (C, D) NK cell-mediated target cell apoptosis leads to loss of ULBP2. 10 5 Jurkat (C) or H9 cells (D) were incubated with (+NK) or without (−NK) in an equal number of IL-2 expanded peripheral blood NK cells at 37°C for 2 hours. The resulting cell mixtures were stained by anti-human ULBP2 or ICAM1/2 antibodies, followed by APC-conjugated goat anti-mouse IgG antibody and Annexin V-PE staining, and then analyzed by flow cytometry. NK cells were excluded by FITC conjugated anti-human CD56 mAb staining.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Incubation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A) Loss of cell surface ULBP2 expression in apoptotic compounds-treated cells. Jurkat cells (upper panels) were treated with 4 µg/ml Actinomycin D (ActD), 4 µM CPT, 25 µM ETO or DMSO for 4 hours in serum-free RPMI 1640 medium, and then were collected for flow cytometry staining. PE-conjugated mouse anti-human ULBP1 and ULBP2 antibodies were used. H9 cells (lower panels) were treated with 4 µg/ml ActD, 4 µM CPT or 50 µM ETO for 12 hours in serum-free RPMI 1640. DMSO-treated cells were used as the control (dotted lines). Biotin-labeled goat anti-human ULBP2 and ULBP3 and PE-conjugated streptavidin were used in this experiment. ULBP1/2/3 expression on control cells and treated cells are shown in dotted lines and solid lines, respectively. Isotype controls are shown in gray-shaded histograms. (B, C) Absence of ULBP2 on Annexin V positive cells. (B) Jurkat cells were treated with ActD for 2 hours in serum-free RPMI 1640 medium. The treated cells were stained by biotin-labeled goat anti-human ULBP1 or ULBP2 polyclonal antibodies, followed by APC-conjugated streptavidin and Annexin V-FITC staining, and then analyzed by flow cytometry. (C) Jurkat cells were treated with ActD for 2 hours, and then ULBP2 and Annexin V staining was visualized by confocal microscopy. Scale bar, 10 µm.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Expressing, Flow Cytometry, Staining, Control, Labeling, Confocal Microscopy

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A) Z-VAD-FMK inhibited apoptotic compound-induced loss of cell surface ULBP2 in Jurkat cells. Jurkat cells were pretreated with 50 µM Z-FA-FMK (dotted lines) or Z-VAD-FMK (solid lines) for 30 min, and then were treated with 2 µg/ml ActD, 4 µM CPT or 20 µM ETO for 4 hours at 37°C. Cell surface expression of ULBP2 was determined by flow cytometric analysis using PE-conjugated mouse anti-human ULBP2. PE-conjugated mouse IgG2a was used as an isotype control (gray-shaded). (B) Z-VAD-FMK inhibited NK cell-mediated loss of cell surface ULBP2 in H9 cells. 1.5×10 5 H9 cells were incubated with IL-2 expanded human primary NK cells at the indicated E:T ratios for 2 hours, and the cell surface expression of ULBP2 was determined by flow cytometric analysis using PE-conjugated mouse anti-human ULBP2 antibody. NK cells were gated out by FSC/SSC and APC-conjugated CD56 antibody. ULBP2 expression in control H9 cells in the absence of NK cells are shown in dotted lines, whereas ULBP2 expression in H9 cells incubated with NK cells are shown in dashed lines (DMSO) and solid lines (Z-VAD-FMK), respectively.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Expressing, Control, Incubation

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A, B) Apoptosis-induced shedding of ULBP2 is more intense than spontaneous shedding of it. (A) 2.5×10 6 Jurkat cells were treated with 2 µg/ml ActD, 4 µM CPT or 20 µM ETO for the indicated time in 0.5 ml serum-free RPMI 1640 medium. (B) 5×10 5 cells H9 were treated with 2 µg/ml Act D, 4 µM CPT or 50 µM ETO for the indicated time in 0.5 ml serum-free RPMI 1640 medium. The supernatant was collected for ELISA assay. DMSO was used as a negative control. (C, D) NK cell-induced shedding of ULBP2. 1×10 5 IL-2 expanded human NK cells were co-cultured with the indicated number of Jurkat (C) or H9 cells (D) for 2 hours, and then culture media were collected for ULBP2 ELISA. (E) Absence of ULBP2 in exosomes. 2×10 7 H9 cells were resuspended in 10 ml serum-free RPMI 1640 medium and treated with ActD or CPT overnight. Exosome preparations from the resulting culture supernatants were used to coat 4-µm-diameter aldehyde/sulfate latex beads by passive adsorption. The coated beads were used for detection of CD63 and ULBP2 by flow cytometric analysis. Beads with exosomes coating are showed in solid lines, and beads without exosomes coating are showed in gray-shaded histograms as controls.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Cell Culture, Adsorption

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A, B) Spontaneous shedding of ULBP2 from Jurkat and H9 cells. Jurkat (A) or H9 (B) cells were cultured with initial seeding cell numbers ranging from 0.5×10 5 to 4×10 5 cells/ml in 48-well plates, the cells were harvested at various time points (from 17 to 98 hours) to achieve various cell densities, and their released ULBP2 in supernatants were determined by ELISA. The expression of ULBP2 was also been determined by FACS using PE-conjugated anti-human ULBP2 antibody. Cell surface expression of ULBP2 in Jurkat and H9 cells are shown in solid lines, and isotype controls are shown in gray-shaded histograms. (C) Shedding of ULBP2 from apoptotic cells. 4×10 6 Jurkat cells were pre-treated with DMSO or 50 µM Z-VAD-FMK for 30 min, and then treated with ActD and CPT for 6 hours in serum free medium. The resulting culture supernatants were collected for ULBP2 ELISA. (D) Z-VAD-FMK fails to block spontaneous shedding of ULBP2. 8×10 4 Jurkat cells were cultured in RPMI 1640 medium with 10% FBS in the presence of 50 µM Z-FA-FMK, Z-VAD-FMK or their carrier control DMSO for the indicated time. The culture supernatants were used to determine ULBP2 concentration.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Blocking Assay, Control, Concentration Assay

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A) BB-94 blocks spontaneous shedding of ULBP2 from Jurkat and H9 cells. 10 6 Jurkat cells or 5×10 5 H9 cells were cultured in the presence of DMSO, Z-VAD-FMK and BB-94 for 24 or 48 hours in RPMI-1640 medium with 10% FBS, the resulting cell culture supernatants were collected for ELISA. (B) BB-94 abrogates NK cell-mediated shedding of ULBP2. 2×10 5 Jurkat or H9 cells were incubated with IL-2 expanded primary human NK cells at the indicated E:T ratios for 4 hours, and the resulting cell culture supernatants were collected for ELISA. (C) BB-94 abrogates apoptotic compound-induced shedding of ULBP2. 4×10 6 Jurkat cells or 4×10 5 H9 cells were treated with ActD or CPT for 6 hours in serum-free RPMI 1640 medium, the culture supernatants were collected to measure ULBP2 concentration by ELISA.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: Natural Killer Cell-Mediated Shedding of ULBP2

doi: 10.1371/journal.pone.0091133

Figure Lengend Snippet: (A, B) BB-94 abrogates apoptotic compound-induced shedding of ULBP2. (A) Jurkat cells were treated with 2 µg/ml Act D, 4 µM CPT or 20 µM ETO for 4 hours in the presence or absence of BB-94. The treated cells were stained by PE-conjugated mouse anti-human ULBP2 antibodies, and then analyzed by flow cytometry. ULBP2 expression on control cells and BB-94 treated cells are shown in dotted lines and solid lines, respectively. PE-conjugated mouse IgG2a was used as an isotype control (gray-shaded). (B) Jurkat cells were treated with ActD for 2 hours in the presence of BB-94, and then ULBP2 and Annexin V staining was visualized by confocal microscopy. Scale bar, 10 µm. (C, D) BB-94 abrogates NK cell-mediated shedding of ULBP2. 10 5 Jurkat (B) or H9 cells (C) were incubated with (+NK) or without (−NK) equal number of IL-2 expanded peripheral blood NK cells at 37°C for 2 hours. The resulting cell mixtures were stained by anti-human ULBP2 or ICAM1/2 antibodies, followed by APC-conjugated goat anti-mouse IgG antibody and Annexin V-PE staining, and then analyzed by flow cytometry. NK cells were excluded by FITC conjugated anti-human CD56 mAb staining.

Article Snippet: The treated cells were stained by mouse anti-human ULBP2 mAb (165903; R&D Systems), followed by DyLight 594 goat anti-mouse IgG (minimal x-reactivity) antibody (BioLegend) staining.

Techniques: Staining, Flow Cytometry, Expressing, Control, Confocal Microscopy, Incubation